Development of a multiplex PCR for rapid molecular serotyping of Haemophilus parasuis
Chaudhuri, Roy R
Williamson, Susanna M
Langford, Paul R
Rycroft, Andrew N
Wren, Brendan W
Haemophilus parasuis molecular serotyping assay
Journal of Clinical Microbiology
American Society for Microbiology
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Howell, K., Maskell, S., Wang, J., Hernandez-Garcia, J., Weinert, L., Luan, S., Chaudhuri, R. R., et al. (2015). Development of a multiplex PCR for rapid molecular serotyping of Haemophilus parasuis. Journal of Clinical Microbiology, 53 3812-3821. https://doi.org/10.1128/JCM.01991-15
Haemophilus parasuis causes Glässer’s disease and pneumonia in pigs. Indirect haemagglutination (IHA) is used to serotype this bacterium, distinguishing fifteen serovars with some non-typeable isolates. The capsule loci of the fifteen reference strains were annotated and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar was identified for all but two non-typeable isolates in our collection of over 200 isolates. Here we describe the development of a multiplex PCR, based on variation within the capsule loci of the fifteen serovars of H. parasuis, for rapid molecular serotyping. The mPCR distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29x10⁵ ng/µl bacterial genomic DNA and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to in silico results. In addition, the two in silico non-typeable isolates were typeable using the mPCR. A further 84 isolates were analysed by mPCR and compared to IHA serotyping results with 90% concordance (excluding those non-typeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA enabling the differentiation of fourteen of the fifteen serovars of H. parasuis.
Haemophilus parasuis, serotyping, capsule, molecular serotyping
This work was supported by a BPEX PhD studentship and a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G003203/1), the UK Department for Environment, Food and Rural Affairs and Zoetis, awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
External DOI: https://doi.org/10.1128/JCM.01991-15
This record's URL: https://www.repository.cam.ac.uk/handle/1810/251123